Cloning, Expression And Evaluation On The Immunogenecity Of Outer Membrane Protein 25 Of Helicobacter Pylori

Backgroud/ObjectiveHelicobacter pylori (H .pylori) infection is the major etiological factor of chronic active gastritis and peptic ulcer. It is also closely associated with the carcinogenesis of gastric tumors including adenocarcinoma and MALT lymphoma. A working group of the International committee on cancer research sponsored by the World Health Organization has categorized H. pylori infection as a class I carcinogen since 1994. Current therapies for eradicating H. pylori infection depend on the combined use of antibiotics. However this strategy has met serious problems such as high cost, low patient compliance, and the increased prevalence of resistant strains. Therefore, the requirement of effective vaccine was urgent, reasonable and practical. Significant but limited progression has been obtained on the study of H. pylori vaccine since 1990. It is reported that H. pylori infection could be prevented or cured through vaccination with low complete eradication rate. Some antigen could even deteriorate gastric mucosal inflammation of host animals. Outer membrane protein (OMP) is a major class of surface protein of H. pylori. Owing to its direct contact with immunesystem of the host, OMPs might be involved in eliciting immunity, anergy or tolerance during the infection. Therefore, the strengthening vaccination of OMP in the presence of mucosal adjuvant might elicit effective protective immunity against infection. In present study, we aimed to evaluate the immunogenicity of OMP25 of H. pylori (HOP25) by cloning and expressing this gene and evaluating its antigenicity in eliciting specific T cell response..Methods1. HOP25 gene was amplified from H. pylori genomic DNA by PCR and inserted into vector pET-22b(+) to generate a recombinant construct pET-22b(+)/HOP25. After sequencing, the predicted composition of amino acid and hydrophobicity of rHOP25 were analyzed by DNAstar5.0 software.2. pET-22b(+)/HOP25 expressing construct was transformed into Exoli BL21(DE3)pLysS and expressed in the presence of IPTG. rHOP25 was purified by Ni-NTA affinity chromatography. The specificity of expressed protein was confirmed by Western-blot.3. Peripheral blood lymphocytes (PBL) were separated from patients with or without H. pylori infection. Proliferation of PBL in response to recombinant HOP25 was evaluated by MTT assay.Results1. HOP25 gene of H. pylori was amplified and inserted into the plasmid pET-22b(+) successfully subsequent to the double digestion of both plasmid and PCR product with EcoR I and Sal I. DNA sequence analysis showed one open reading frame of 2085bp, which coded a polypeptides with 695 amino acids. The molecular mass was approximate 72.4kDa as predicted by DNA star5.0 software with goodantigenecity and hydrophobicity. The similarity between HOP25 gene we cloned and that of H. pylori strain (ATCC26695) presented in GeneBank was 99.7% (2078/2085).2. SDS-PAGE showed the inducible expression of a protein with molecular weight of about 72kDa. The portion of soluble expressing recombinant protein was over 18% of that of total cell wet weight. The purity of rHOP25 was over 96% after purification by Ni-NTA resin affinity chromatography. Western blot showed it could be recognized by the monoclonal antibody against HOP25.3. rHOP25 can only stimulate proliferation of T cell that isolated from patients with H. pylori infection. Significant difference was found between proliferation of T cells isolated from H. pylori infected and uninfected subjects (P>0.05).Conclusions1. The expressing constructs pET-22b(+)/HOP25have been generated by genetic engineering techniques. Large amount and high purity recombinant HOP25 of H. pylori was produced after transformation into E. coli and purified by Ni-NTA resin column.2. rHOP25 is able to stimulate T cell proliferation. It indicated that HOP 25 specific T cell clones have been induced in blood of subjects with H. pylori infection, thus, suggesting HOP25 might be a putative immunogen in the context of the development of H. pylori vaccine.